The term "reverse transcriptase" describes a class of polymerases characterized as RNA dependent DNA polymerases. All known reverse transcriptases require a primer to synthesize a DNA transcript from an RNA template. Historically, reverse transcriptase has been used primarily to transcribe mRNA into cDNA which can then be cloned into a vector for further manipulation.
Avian myoblastosis virus (AMV) reverse transcriptase was the first widely used RNA dependent DNA polymerase (Verma, 1977, Biochem. Biophys. Acta 473:1). The enzyme has both a 5'-3' RNA directed DNA polymerase activity as well as an RNAseH activity. RNAseH is a processive 5' and 3' riboexonuclease specific for the RNA strand of RNA-DNA hybrids (Perbal, 1984, A Practical Guide to Molecular Cloning, Wiley & Sons New York). Errors in transcription cannot be corrected by reverse transcriptase (Saunders and Saunders, 1987, Microbial Genetics Allied to Biotechnology, Croom Helm, London). A detailed study of the activity of AMV reverse transcriptase and its associated ribonuclease H has been presented by Berger et al., 1983, Biochemistry 22:2365-2372.
Berger et al. found that the rate limiting step in RNA transcription was initiation of the transcription reaction, rather than the sequential polymerization of additional nucleotides. To overcome this limitation, use of a stoichiometric, rather than catalytic, quantity of reverse transcriptase is frequently recommended (Buell et al., 1978, J. Biol. Chem. 253:2471-2482; Wickens et al., 1978, J. Bio. Chem. 253:2483-2495; Yoo et al., 1982, Proc. Nat. Acad. Sci. USA 80:1194-1198; and Okayama and Berg, 1982, Mol. Cell. Biol. 2:161-170). However, when stoichiometric amounts of reverse transcriptase are used, the low level of RNAseH activity is significant and may be responsible for fragmented cDNAs and limited cDNA yields. Christopher et al., 1980, Eur. J. Biochem. 111:4190-4231, and Michelson et al., 1983, Proc. Nat. Acad. Sci. USA 80:472-476, have suggested that including an RNAse inhibitor in cDNA reactions could alleviate this problem.
DNA polymerases isolated from mesophilic microorganisms such as E. coli. have been extensively researched (see, for example, Bessman et al., 1957, J. Biol. Chem. 233:171-177 and Buttin and Kornberg, 1966, J. Biol. Chem. 241:5419-5427). E. coli DNA polymerase I (Pol I) is useful for a number of applications including: nick-translation reactions, DNA sequencing, in vitro mutagenesis, second strand cDNA synthesis, polymerase chain reactions (PCR), and blunt end formation for linker ligation (Maniatis et al., 1982, Molecular Cloning: A Laboratory Manual Cold Spring Harbor, N.Y.).
Several laboratories have shown that some DNA polymerases are capable of in vitro reverse transcription of RNA (Karkas, 1973, Proc. Nat. Acad. Sci. USA 70:3834-3838; Gulati et al., 1974, Proc. Nat. Acad. Sci. USA 71:1035-1039; and Wittig and Wittig, 1978, Nuc. Acid Res. 5:1165-1178). Gulati et al. found that E. coli Pol I could be used to transcribe Q.beta. viral RNA using oligo(dT).sub.10 as a primer. Wittig and Wittig have shown that E. coli Pol I can be used to reverse transcribe tRNA that has been enzymatically elongated with oligo(dA). However, as Gulati et al. demonstrated, the amount of enzyme required and the small size of the cDNA product suggests that the reverse transcriptase activity of E. coli Pol I has little practical value.
The thermostable DNA polymerase from Thermus aquaticus (Taq) has been cloned, expressed, and purified from recombinant cells (Lawyer et al., 1989, J. Biol. Chem. 264:6427-6437 and European patent publication EP 258,017, incorporated herein by reference). Crude preparations of a DNA polymerase activity isolated from T. aquaticus have been described by others (Chien et al., 1976, J. Bacteriol. 127:1550-1557, and Kaledin et al., 1980, Biokymiya 45:644-651).
Taq polymerase, a 94 kDa enzyme, has a 75.degree. C. temperature optimum activity. The enzyme is not permanently inactivated even when heated to 93.degree.-95.degree. C. for brief periods of time, as, for example, in the practice of DNA amplification by the polymerase chain reaction (PCR). In contrast, at this elevated temperature E. coli DNA Pol I and reverse transcriptases are inactivated.
Like E. coli Pol I, Taq polymerase requires a primer for initiation of synthesis; however, the thermostable properties of Taq are advantageous for extension of a DNA primer on a DNA template. Enhanced specificity of primer binding at elevated temperatures results in a higher yield of the desired product with less nonspecific product. When used in PCR, Taq needs to be added only at the beginning of the reaction rather than before each round of amplification, as is necessary when using E. coli Pol I. PCR methods for amplifying and detecting DNA sequences are disclosed in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159, incorporated herein by reference.
PCR requires a nucleic acid template and appropriate primers for amplification. The DNA to be amplified may be synthetic or genomic, contained in a plasmid, or contained in a heterogenous sample. If the nucleotide sequence to be amplified is RNA, the nucleic acid molecule is first treated with reverse transcriptase in the presence of a primer to provide a cDNA template for amplification. Prior to the present invention, amplification of RNA necessitated a reverse transcription step with, e.g., non-thermostable reverse transcriptases such as AMVRT, followed by treatment of the resulting cDNA with a DNA polymerase. The amplification of RNA could be greatly simplified by the availability of a method for reverse transcribing RNA and amplifying DNA with a single enzyme.
The present invention addresses this need. The present invention provides methods for the efficient amplification of RNA sequences requiring only one enzyme, a thermostable DNA polymerase. These methods offer simplicity and enhanced specificity over currently known methods.